Transplantation of human neural cells for treatment of neurodegenerative conditions

ABSTRACT

A method of treating neurodegenerative conditions is provided. Neural stem cells may be implanted at and/or remote from a region of neuron degeneration. The methods can include isolating neural stem cells from regions where specific types of neurons corresponding to the neurons to be replaced are generated. The methods can include isolating neural stem cells secreting growth factors affecting the growth and/or regeneration of specific types of neuron. In this invention, we disclose a method of treating such disorders, including several neurodegenerative disorders arising from the lack of cells that produce particular neurotransmitters in neural circuitry by transplanting exogenously cultured and expanded neural progenitors which, upon transplantation into a neural tissue, differentiate into neurons capable of integrating and producing neurotransmitters in sufficient quantities and in a sufficient manner to overcome the symptoms associated with the neurodegeneration.

PRIORITY CLAIM

The present application claims the benefit of U.S. Provisional PatentApplication 60/629,220, filed Nov. 17, 2004, which is incorporatedherein in its entirety by reference.

This work was supported by grants from the United States Governmentfunded through the National Institutes of Health. The U.S. Governmenthas certain rights in this invention.

BACKGROUND

The disclosed methods relate to methods of treating disorders throughtransplantation of cells that are uniquely beneficial for such treatmentmethods. In particular, the disclosed methods provide methods oftreating neurodegenerative conditions with neural stem cells (NSCs).

Neurodegenerative disorders are characterized by conditions involvingthe deterioration of neurons as a result of disease, hereditaryconditions or injury, such as traumatic or ischemic spinal cord or braininjury.

The circuitry of the spinal cord that governs contraction of skeletalmuscles of the limbs, involves excitatory motor neurons and inhibitoryGABAergic (i.e., GABA-producing) and glycinergic (i.e.,glycine-producing) inter-neurons. A motor neuron is a nerve thatoriginates from the anterior horn of the gray matter of the spinal cord.The axon of the motor neuron emerges from a segment of the spinal cordas an efferent motor fiber that innervates muscle fibers. Impulsesconducted by the motor neuron stimulate the muscle fibers to contract.GABA, gamma-amino butyric acid, is a naturally occurring metabolite ofthe mammalian nervous system which acts as a neurotransmitter to inhibitor dampen the nerve conduction of electrical potential. Loss ofGABAergic interneurons results in dysregulation of the inhibitorytonality of the motor neuron-evoked muscle contractions. Without thecontrol exerted by inhibitory interneurons on excitatory neurons, anover-firing of excitatory neurons occurs leading to spastic uncontrolledcontraction or uncontrolled rigidity of the muscles of the limbs. Lossof motor neurons results in flaccid paraplegia in which the subjectscannot contract the muscles and are thereby unable to move.

One instance in which GABAergic interneurons are damaged in the spinalcord includes a complication associated with a transient cross-clampingof the descending thoracic or thoracoabdominal aorta. Suchcross-clamping is a necessary step in vascular surgery to repairaneurysms of thoracic or thoracoabdominal aorta. For the duration of thecross-clamping, a portion of the spinal cord does not receive bloodcirculation and can become ischemic. Depending on the duration of theischemic interval, subsequent neurodegenerative dysfunction may beexpressed neurodegeneratively as paraparesis or fully-developed spasticor flaccid paraplegia.

While the mechanism leading to ischemia-induced neuronal degeneration isonly partially understood and may involve excessive release/activity ofexcitatory amino acids, prostaglandins and/or oxygen free radicals, theneuronal population of spinal cord affected by transient ischemic insultare well defined. For example, histopathological analysis of spinal cordtaken from animals with fully developed spastic paraplegia shows aselective loss of small inhibitory neurons; however, alpha-motoneuronspersist in previously ischemic spinal segments. Similar spinal neuronalpathology in human subjects having spinal ischemic injury has beendescribed.

In contrast, in animals with flaccid paraplegia, pan-necroticneurodegenerative changes are seen affecting both small inhibitory andexcitatory interneurons as well as ventral motor neurons. During theperiod of neuronal degeneration after spinal ischemia, aninjury-dependent activation of local microglia and inflammatory changes,such as infiltration with macrophages, is also seen as in focal orglobal brain ischemia. Depending on the extent of injury, theinflammatory changes typically peak between two to seven days afterischemic insult and then show gradual loss of inflammatory elements overtwo to four weeks of post-ischemic period.

In the past two to three decades, a considerable effort has been made toassess in animal models the therapeutic potential of spinal grafting ofa variety of materials. Thus, cell lines or acutely isolated spinal cordfetal tissue have been delivered to injured regions and direct spinalgene therapy has also been used to ameliorate neurodegenerativedysfunctions in several models of spinal injury, including mechanicaltraumatic injury, chemically lesioned spinal cord or geneticallymanipulated animals with progressive α-motoneuronal degeneration (ALStransgenic mice or rat).

In general, studies demonstrate long-term survival and preservation ofneuronal phenotypes in grafts generated from fetal tissue, but not fromneural precursors that have been expanded in vitro. In fact, onlylimited neuronal differentiation and maturation of neural precursorsexpanded in vitro and grafted into mechanically or chemically injuredspinal cord has been demonstrated. Cells preferentially differentiateinto non-neuronal cell types. While the mechanism of this preferentialnon-neuronal differentiation is not completely understood, it ishypothesized that a local release of pro-inflammatory cytokines (such asTNFα, TGFβ at the site of previous injury is likely involved.

Neurodegeneration represents a particularly challenging biologicalenvironment for cell therapy and cell death signals present inestablished neurodegenerative disease (Rothstein et al., 1992; Howlandet al., 2002; Turner et al., 2005) may be incompatible with graftsurvival. In addition, the adult spinal cord is viewed as lacking cellsand/or signals allowing regeneration (Park et al., 2002), and themajority of NSC grafting studies have shown poor or restricteddifferentiation (Cao et al., 2002; Yan et al., 2003; Yan et al., 2004).

One of the major problems in cell therapeutics is low cell survival(less than 5%) of the cells grafted. All of the grafted cells to dateundergo significant cell death shortly after injection in vivo. Thus, inorder to deliver an effective dose of cells, the final dose must beinjected at least 20 times. This, in turn, requires a much larger scaleof cell manufacturing which poses further regulatory and economicobstacles. Furthermore, the survival rate of such cells in vivo has notbeen able to be maintained. Failure to demonstrate reproducibleadministration of effective doses of cell therapy prevents approval foruse by government and other regulatory agencies such as the Food andDrug Administration.

Additional challenges are presented when treating neurodegenerativediseases and conditions that are disseminated over a large area of abody, tissue, or organ, such as the entire nervous system rather than asingle localized area. For example, in ALS, neurodegeneration involvesslow death of motor neurons along the entire spinal cord as well asthose neurons in motor cortex. Likewise, in most lysosomal diseases,neuronal destruction involves most regions of the brain and spinal cord.Alzheimer's disease involves most of the cerebrum. Even in morelocalized neurodegenerative diseases such as Parkinson's andHuntington's, the affected area of striatum is quite large, much largerthan the grafting area that can be surgically reached. Thus, celltherapeutics for neurodegenerative diseases are expected to requirewider grafting procedures.

There is, therefore, a need for improved methods of treatingneurodegenerative conditions. There is also a need for improved methodsof culturing and transplanting human neural stem cells and human neuralprogenitors that once grafted overcome all of the previously seenlimitations and provide functional benefit. Thus, this method oftreating neurodegenerative conditions, in vivo, generates robustneuronal differentiation, permits long-term neuronal survival undervarious degenerative conditions and maturation into therapeuticallyrelevant subpopulations of neurons in adult tissues that lackdevelopmental cues, and provides wide therapeutic range than thelocation of the cells themselves.

SUMMARY

The disclosed methods include methods for treating neurodegenerativeconditions. In particular, the disclosed methods include transplantinginto a subject in need thereof NSCs, neural progenitors, or neuralprecursors that have been expanded in vitro such that the cells canameliorate the neurodegenerative condition. In an embodiment, thedisclosed methods include identifying, isolating, expanding, andpreparing the donor cells to be used as treatment of theneurodegenerative condition. The donor cells to be transplanted can beselected to correspond to the elements or lack thereof that contributeto the condition, its symptoms and/or its effects.

The cells of the disclosed methods include cells that, upontransplantation, generate an amount of neurons sufficient to integratewithin the neuronal infrastructure to ameliorate a disease state orcondition. In an embodiment, the disclosed methods include treatingneurodegenerative diseases or conditions by transplanting multipotentialneural progenitors or neural stem cells isolated from the centralnervous system of a mammal and that have been expanded in vitro. Forexample, transplantation of the expanded neural stem cells can be usedto improve ambulatory function in a subject suffering from various formsof myelopathy with symptoms of spasticity, rigidity, seizures, paralysisor any other hyperactivity of muscles.

A method of treatment can include supplying to an injured neural area,via transplantation, a suitable number of NSCs which can differentiateinto a sufficient number of GABA-producing neurons and/orglycine-producing neurons to attenuate defective neural circuits,including hyperactive neural circuits.

In an embodiment, the disclosed methods include restoring motor functionin a motor neuron disease. A suitable number or a therapeuticallyeffective amount of NSCs or neural progenitors which are capable ofdifferentiating into motor neurons can be provided to at least one areaof neurodegeneration, such as a degenerative spinal cord, to restoremotor function. The NSCs exert their therapeutic effect by replacingdegenerated neuromuscular junctions.

In conjunction or alternatively, the NSCs exert their therapeutic effectby expressing and releasing trophic molecules which protect the neuronsof the degenerating tissue so that more of them survive for longerperiod of time. NSC-derived neurons can be prompted to project intoventral roots and innervate muscle where the NSCs engage in extensivereciprocal connections with host motor neurons in subjects withdegenerative motor neuron disease. Therefore, in an embodiment, NSCsfrom human fetal spinal cord can be grafted into the lumbar cord wherethese cells can undergo differentiation into neurons that form synapticcontacts with host neurons and express and release motor neuron growthfactors.

In an embodiment, the disclosed methods include providing neural stemcells or neural progenitors that integrate with the host tissue andprovide one or more growth factors to the host neurons therebyprotecting them from degenerative influences present in the tissue. Themethods include introducing a sufficient number of NSCs or neuralprogenitors to an area of a spinal cord such that an effective amount ofat least one growth factor is secreted by the NSCs.

In an embodiment, the disclosed methods include providing a method forusing animal models in the preclinical evaluation of stem cells for cellreplacement in neurodegenerative conditions.

In an embodiment, the disclosed methods include increasingdifferentiation efficiency of transplanted NSCs into neurons. The methodincludes expanding highly enriched NSCs or neural progenitors in theirundifferentiated state so that, upon transplantation, a sufficientnumber such as 20% of the cells in the graft adopts a neuronal fate.

In an embodiment, the disclosed methods include increasing the number ofdifferentiated cells without increasing the number of NSCs or neuralprogenitors to be transplanted. In an embodiment, the method includespreparing the expanded donor population in such a way that, oncetransplanted, the NSCs or neural progenitors continue to divide in vivoas many as ten times and without generating a tumor, thereby,effectively increasing the total number of delivered cells.

The cells of the disclosed methods can be isolated or obtained fromfetal, neonatal, juvenile, adult, or post-mortem tissues of a mammal.The cells of the disclosed methods can be isolated or obtained from thecentral nervous system, blood, or any other suitable source of stemcells that differentiate into neurons. The cells can also be obtainedfrom embryonic stem cells. For instance, in an embodiment, the cellsinclude neuroepithelial cells isolated from the developing fetal spinalcord. In certain instances, the neural precursor cells can be neuralprogenitors isolated from specific sub-regions of the central nervoussystem.

According to the disclosed methods the neural stem cells are expanded inculture. In an embodiment, the neural precursor cells can bemultipotential NSCs capable of expansion in culture and of generatingboth neurons and glia upon differentiation.

The cells can be either undifferentiated, pre-differentiated or fullydifferentiated in vitro at the time of transplantation. In an embodimentthe cells are induced to differentiate into neural lineage. The cells ofthe disclosed methods can undergo neuronal differentiation in situ inthe presence of pro-inflammatory cytokines and other environmentalfactors existing in an injured tissue.

Using the present methods, neural circuits can be treated bytransplanting or introducing the cells into appropriate regions foramelioration of the disease, disorder, or condition. Generally,transplantation occurs into nervous tissue or non-neural tissues thatsupport survival of the grafted cells. NSC grafts employed in thedisclosed methods survive well in a neurodegenerative environment wherethe NSCs can exert powerful clinical effects in the form of delaying theonset and progression of neurodegenerative conditions or disease.

In some instances, transplantation can occur into remote areas of thebody and the cells can migrate to their intended target. Accordingly,the disclosed methods can also include partial grafting of human NSCs.As used herein, the term “partial grafting” can refer to theimplantation of expanded NSCs in only a portion of an area or less thanan entire area of neurodegeneration. For example, partial grafting ofhuman NSCs into the lumbar segments of spinal cord. At least a portionof the effects of NSCs on degenerating motor neurons include delivery ofneurotrophins and trophic cytokines to degenerating host motor neuronsvia classical cellular mechanisms. To this end, NSCs undergoing partialgrafting into the lumbar segments of spinal cord using the disclosedmethods have been shown in a transgenic animal model of motor neurondisease to survive, undergo extensive neuronal differentiation, promotemotor neuron survival and function in the immediate area oftransplantation as well as areas remote from the area oftransplantation.

Accordingly, the disclosed methods provide a method of treatingspasticity, rigidity, or muscular hyperactivity conditions. The methodincludes isolating at least one neural stem cell from a mammal andexpanding in vitro the neural stem cell to an expanded population. Themethod also includes concentrating the expanded population andintroducing a therapeutically effective amount of the expandedpopulation to at least one area of a recipient spinal cord. At least 20%of the expanded population is capable of generating neurons in therecipient spinal cord.

In an embodiment, the conditions derive from traumatic spinal cordinjury, ischemic spinal cord injury, traumatic brain injury, stroke,multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease,amyotropic lateral sclerosis, chronic ischemia, hereditary conditions,or any combination thereof.

In an embodiment, the neural stem cell is isolated from a sourceselected from the group consisting of a central nervous system, aperipheral nervous system, bone marrow, peripheral blood, umbilical cordblood and at least one embryo.

In an embodiment, the mammal is a developing mammal.

In an embodiment, the gestational age of the developing mammal isbetween about 6.5 to about 20 weeks.

In an embodiment, the neural stem cell is isolated from a human fetalspinal cord.

In an embodiment, expanding the neural stem cell includes culturing theneural stem cell in the absence of serum.

In an embodiment, expanding the neural stem cell includes exposing theneural stem cell to at least one growth factor.

In an embodiment, the growth factor is selected from the groupconsisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.

In an embodiment, the therapeutically effective amount of the expandedpopulation is capable of generating at least 1,000 GABA-producingneurons in vivo.

In an embodiment, the therapeutically effective amount of the expandedpopulation is capable of generating at least 1,000 glycine-producingneurons in vivo.

In an embodiment, at least 40% of the expanded population is capable ofgenerating neurons in the spinal cord.

In an embodiment, introducing the therapeutically effective amount ofthe expanded population includes injecting at least a portion of thetherapeutically effective amount into a plurality of areas of therecipient spinal cord.

In an embodiment, at least 30% of the expanded population is capable ofdifferentiating into neurons in vitro.

In another embodiment a neural stem cell is provided. The neural stemcell is capable of treating spasticity, rigidity or muscularhyperactivity conditions. The neural stem cell is isolated from a mammaland expanded in vitro to an expanded population. The expanded populationincluding the stem cell is concentrated and a therapeutically effectiveamount of the expanded population is introduced to at least one area ofa recipient spinal cord. At least 20% of the expanded population iscapable of generating neurons in the recipient spinal cord.

In another embodiment of the disclosed methods, a method of treatingchronic pain is provided. The method includes isolating at least oneneural stem cell from a mammal and expanding in vitro the neural stemcell to an expanded population. The method also includes concentratingthe expanded population and introducing a therapeutically effectiveamount of the expanded population to at least one area of a recipientspinal cord. At least 20% of the expanded population is capable ofgenerating neurons in the recipient spinal cord.

In an embodiment, the chronic pain derives from traumatic spinal cordinjury, ischemic spinal cord injury, traumatic brain injury, stroke,multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease,amyotropic lateral sclerosis, chronic ischemia, hereditary conditions,or any combination thereof.

In an embodiment, the therapeutically effective amount of the expandedpopulation is capable of generating at least 1,000 GABA-producingneurons.

In an embodiment, the therapeutically effective amount of the expandedpopulation is capable of generating at least 1,000 glycine-producingneurons.

In an embodiment, at least 40% of the expanded population is capable ofgenerating neurons in the spinal cord.

In an embodiment, introducing the therapeutically effective amount ofthe expanded population includes injecting at least a portion of thetherapeutically effective amount into a plurality of areas of therecipient spinal cord.

In an embodiment, the areas include doral horn.

In an embodiment, the areas include intrathecal space.

In a further embodiment a neural stem cell is provided. The neural stemcell is capable of treating chronic pain. The neural stem cell isisolated from a mammal and expanded in vitro to an expanded population.The expanded population including the stem cell is concentrated and atherapeutically effective amount of the expanded population isintroduced to at least one area of a recipient spinal cord. At least 20%of the expanded population is capable of generating neurons in therecipient spinal cord.

In another embodiment of the disclosed methods, a method of treatingmotor neuron degeneration is provided. The method includes isolating atleast one neural stem cell from a mammal and expanding in vitro theneural stem cell to an expanded population. The method also includesconcentrating the expanded population and introducing a therapeuticallyeffective amount of the expanded population to at least one area of arecipient spinal cord. At least 20% of the expanded population iscapable of generating neurons in the recipient spinal cord.

In an embodiment, the motor neuron degeneration derives from traumaticspinal cord injury, ischemic spinal cord injury, traumatic brain injury,stroke, multiple sclerosis, cerebral palsy, epilepsy, Huntington'sdisease, amyotropic lateral sclerosis, chronic ischemia, hereditaryconditions, or any combination thereof.

In an embodiment, the method includes isolating the neural stem cellfrom an area rich in at least one neuronal subtype, wherein the neuronalsubtype produces a growth factor effective in ameliorating the motordeficit.

In an embodiment, the expanded population includes an amount of neuralstem cells capable of differentiating into neurons sufficient to secretea therapeutically effective amount of at least one growth factor.

In an embodiment, the method includes isolating the neural stem cellfrom an area rich in motor neurons.

In a further embodiment a neural stem cell capable of treatingsyringomyelia is provided. The neural stem cell is isolated from amammal and expanded in vitro to an expanded population. The expandedpopulation including the stem cell is concentrated and a therapeuticallyeffective amount of the expanded population is introduced to at leastone area of a recipient spinal cord. At least 20% of the expandedpopulation is capable of generating neurons in the recipient spinalcord.

In another embodiment of the disclosed methods, a method of treatingsynngomyelia is provided. The method includes isolating at least oneneural stem cell from a mammal and expanding in vitro the neural stemcell to an expanded population. The method also includes concentratingthe expanded population and introducing a therapeutically effectiveamount of the expanded population to a syrinx of a recipient spinalcord. At least 20% of the expanded population is capable of generatingneurons in the syrinx of the recipient spinal cord.

In an embodiment, the syringomyelia derives from traumatic spinal cordinjury, ischemic spinal cord injury, traumatic brain injury, stroke,multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease,amyotropic lateral sclerosis, chronic ischemia, hereditary conditions,or any combination thereof.

In an embodiment, the method includes isolating the neural stem cellfrom an area rich in at least one neuronal subtype, wherein the neuronalsubtype produces a growth factor effective in ameliorating thesyringomyelia.

In an embodiment, includes isolating the neural stem cell from an arearich in motor neurons.

In an embodiment, the expanded population includes an amount of neuralstem cells capable of differentiating into neurons sufficient to secretea therapeutically effective amount of at least one growth factor.

In an embodiment, the therapeutically effective amount of the expandedpopulation is capable of generating at least 1,000 neurons.

In an embodiment, at least 100,000 neural stem cells of the expandedpopulation are introduced to the syrinx of the recipient spinal cord.

In yet a further embodiment, a neural stem cell capable of treatingsyringomyelia is provided The neural stem cell is isolated from a mammaland expanded in vitro to an expanded population. The expanded populationincluding the stem cell is concentrated and a therapeutically effectiveamount of the expanded population is introduced to a syrinx of arecipient spinal cord. At least 20% of the expanded population iscapable of generating neurons in syrinx the recipient spinal cord.

In an additional embodiment of the disclosed methods, a method ofexpanding in vitro at least one neural stem cell to an expandedpopulation of neural stem cells is provided. Each neural stem cellexpansion exceeds thirty cell doublings without differentiating. Themethod includes dissociating neural stem cells from central nervoussystem tissue and providing at least one extracellular protein to aculture vessel. The extracellular protein includes at least about 10μg/mL of poly-D-lysine and about 1 mg/ml fibronectin. The method alsoincludes culturing the dissociated neural stem cells in the culturevessel in the absence of serum and adding to the culture vessel at leastone growth factor. The growth factor is selected from the groupconsisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof. Themethod further includes passaging the cultured cells prior toconfluence.

In an embodiment, the expanded neural stem cells are capable ofdifferentiating into neurons.

In an embodiment, expanding the neural stem cell includes addingfibronectin to culture medium as a soluble factor.

In an embodiment, dissociating the cells and passaging the cellsincludes enzymatic dissociation.

In an embodiment, the enzymatic dissociation includes treating the cellswith trypsin.

In an embodiment, a therapeutically effective amount of the expandedpopulation is introduced to at least one area of a recipient nervoussystem to treat a neurodegenerative condition.

It is therefore an advantage of the disclosed methods over existingpharmacological strategies to provide a method of facilitating theability of the transplanted NSCs to secrete trophic molecules which canbe delivered to degenerating motor neurons under conditions of optimalbiovailability.

Yet another advantage of the present invention is to provide a method ofculturing and expanding NSCs from human fetal spinal cord to facilitatethe successful engraftment of the NSCs into the lumbar cord.

A further advantage of the disclosed methods includes providing a methodof achieving a higher proportion of neuronal differentiation of apopulation of NSCs.

Another advantage of the disclosed method includes achieving clinicaleffects from partial grafting of NSCs.

Additional features and advantages of the disclosed methods aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Expansion of human spinal stem cells. A human spinal progenitor(a.k.a. NSC) line was isolated from a 7-8 week old post-mortem fetalspinal cord tissue and serially passaged for about 130 days of netculture period. At each passage, the cell number recovered at harvestwas divided by the initial cell number at plating to obtain thefold-increase in cell number. Cumulative fold-increase (left Y axis) wasobtained by multiplying the fold-increase at each passage. Doubling time(right Y axis) of the cells at each passage was calculated by dividingthe fold-increase in cell number by the each culture period (X axis).This process was repeated three times (serial expansion 1, 2, and 3).

FIG. 2. Morphology of expanded human spinal stem cells. (A) Phasecontrast view of a fixed, unstained, expanding culture, 20× objective,(B) Anti-nestin antibody staining.

FIG. 3. Characterization of differentiated cultures obtained fromexpanded human spinal stem cells. The expanded cells of passage 15-16were differentiated for approximately 14 days in culture, fixed, andstained with various neuron-specific antibodies. (A) Tau and MAP2; (B)Type 3 beta tubulin; (C) GABA; (D) Acetylcholine transferase.

FIG. 4. Expansion of human midbrain stem cells. A human midbrainprogenitor (a.k.a. NSC) line was isolated from a 7-8 week oldpost-mortem fetal mid brain tissue and serially passaged for about 170days of net culture period. At each passage, the cell number recoveredat harvest was divided by the initial cell number at plating to obtainthe fold-increase in cell number. Cumulative fold-increase (Y axis) wasobtained by multiplying the fold-increase at each passage.

FIG. 5. Dopamine uptake activity of expanded human midbrain stem cells.Dopamine trasporter activity (DAT) in live cells was determined from ahuman midbrain stem cell line and one of its clonal subline, which weredifferentiated for 22 or 44 days at the time of assay. The cells wereincubated with radiolabeled dopamine in the presence (+) or absence (−)of the DAT inhibitor nomifensine (10 μM). Cells were washed to removeunincorporated dopamine and lysed in a scintillation cocktail. The totalcellular radioactivity (dpm) was then determined using a scintillationcounter.

FIG. 6. Effect of exogenous factors on induction of neuronaldifferentiation and dopaminergic differentiation of human midbrain stemcell lines. Cryopreserved neural stem cells from two human midbrain stemcell lines (527RMB and 796RMB) were thawed and plated at a density of40,000 cells per well in 4-well chamber slides in the presence of bFGFand allowed to proliferate for 6 days. Subsequently, bFGF was removedand cells were allowed to differentiate for additional 8 days. Cellswere divided into four groups based on the timing and duration ofexposure to sertoli cell conditioned medium (SCCM, diluted 1:1 in N2).One group was exposed to SCCM during proliferation and differentiation(condition 1); a second was exposed during proliferation only (condition2); a third was exposed during differentiation only (condition 3); and afourth was not exposed to SCCM (Control, Cont.). Media was changed everyother day, and mitogen was added daily during the proliferative phase.Four wells were maintained per condition to allow staining for multiplemarkers. Upon differentiation, the cells were fixed using 4%paraformaldehyde and immunostained using antibodies to MAP2ab (FIG. 6A)and tyrosine hydroxylase (FIG. 6B), as well as GFAP and GalC.Immunostained cells were counted using a 40× objective, and at leastthree fields were counted for each well. Few or no GFAP+ or GalC+ cellswere detected upon analysis of cells maintained under any condition, sothese antigens were excluded from the analysis.

FIG. 7. Reduction of spasticity/rigidity and motor deficits in rats byhuman spinal stem cell transplantation. Spastic rats were produced byischemic lesioning of the lumbar spinal cord. In one group (blackcircle), the rats (n=9) were transplanted with human spinal stem cellsexpanded in culture (passage 16), while the other, control, group (whitecircle, n=7) received only the media without the cells. Theimmunosuppressant, FK506, was administrated at 1 mg/kg daily to bothgroups for the duration of the study (8 weeks). The motor coordinationof individual animals was assessed by BBB scoring once every week.

FIG. 8. Reduction of spasticity/rigidity and motor deficits in rats byhuman spinal stem cell transplantation. Spastic rats were produced byischemic lesioning of the lumbar spinal cord. In one group (black circleand black squire), the rats (n=13) were transplanted with human spinalstem cells expanded in culture (passage 16), while the other, control,group (filled triangle, n=6) received only the media without the cells.The immunosuppressant, FK506, was administrated at 3 mg/kg daily to bothgroups for the duration of the study (12 weeks). The motor coordinationof individual animals was assessed by BBB scoring once every week.

FIG. 9. Effects of human NSC treatment on severity of motor neurondisease in G93A SOD1 rats shown with progression (A-B) as well asend-point (C-E) analysis of clinical and pathological measures in caseswith live-cell (L, red) and dead-cell (control, C) grafts (blue).

A-B. Panel A is a Kaplan-Meier plot showing a significant separationbetween experimental and control animals throughout the course ofobservation (P=0.0003). Panel B shows a separation in the two principalmeasures of muscle weakness (BBB and incline plane scores) between thetwo groups (P=0.00168 and 0.00125, respectively).

C-E. End-point analysis of survival (C), time-to-disease-onset (D) andmotor neuron numbers (E) in experimental and control rats. Panel C showsa significant 11-day difference in life span between the two groups(P=0.0005). Panel D shows a significant 7-day difference intime-to-disease-onset between the two groups (P=0.0001). Panel E shows adifference of 3, 212 cells in the lumbar protuberance between live anddead NSC groups (P=0.01). Inset at the bottom of (E) illustrates thedifference in motor neuron survival between a representativeexperimental (up) and control (down) rat at 128 days of age; arrowsindicate the lateral motor neuron group. Size bars: 150 μm.

DETAILED DESCRIPTION

The disclosed methods are related to treating neurodegenerativeconditions. In particular, the disclosed methods include methods ofpreparing neural stem cells for transplantation into a subject in needthereof. Preparing the cells for transplantation can include expandingin vitro a specific population of cells to a level sufficient forcommercial use as a treatment for neurodegenerative conditions. In anembodiment, the method of treatment of a degenerated or an injuredneural area includes supplying to the area an effective number of neuralstem cells sufficient to ameliorate the neurodegenerative condition.

As used herein, a neurodegenerative condition can include any Disease ordisorder or symptoms or causes or effects thereof involving the damageor deterioration of neurons. Neurodegenerative conditions can include,but are not limited to, Alexander Disease, Alper's Disease, AlzheimerDisease, Amyotrophic Lateral Sclerosis, Ataxia Telangiectasia, CanavanDisease, Cockayne Syndrome, Corticobasal Degeneration, Creutzfeldt-JakobDisease, Huntington Disease, Kennedy's Disease, Krabbe Disease, LewyBody Dementia, Machado-Joseph Disease, Multiple Sclerosis, ParkinsonDisease, Pelizaeus-Merzbacher Disease, Niemann-Pick's Disease, PrimaryLateral Sclerosis, Refsum's Disease, Sandhoff Disease, Schilder'sDisease, Steele-Richardson-Olszewski Disease, Tabes Dorsalis or anyother condition associated with damaged neurons. Other neurodegenerativeconditions can include or be caused by traumatic spinal cord injury,ischemic spinal cord injury, stroke, traumatic brain injury, andhereditary conditions.

The disclosed methods include the use of NSCs to ameliorate aneurodegenerative condition. As used herein, the term, “NSCs” can alsorefer to neural or neuronal progenitors, or neuroepithelial precursors.NSCs can be functionally defined according to their capacity todifferentiate into each of the three major cell types of the CNS:neurons, astrocytes, and oligodendrocytes.

In an embodiment, the NSCs are multipotential such that each cell hasthe capacity to differentiate into a neuron, astrocyte oroligodendrocyte. In an embodiment, the NSCs are bipotential such thateach cell has the capacity to differentiate into two of the three celltypes of the CNS. In an embodiment, the NSCs include at leastbipotential cells generating both neurons and astrocytes in vitro andinclude at least unipotential cells generating neurons in vivo.

Growth conditions can influence the differentiation direction of thecells toward one cell type or another, indicating that the cells are notcommitted toward a single lineage. In culture conditions that favorneuronal differentiation, cells, particularly from human CNS, arelargely bipotential for neurons and astrocytes and differentiation intooligodendrocytes is minimal. Thus, the differentiated cell cultures ofthe disclosed methods may give rise to neurons and astrocytes. In anembodiment, the ratio of neurons to astrocytes can approach a 50:50ratio.

The disclosed methods include obtaining NSCs residing in regions of amammalian CNS such as the neuroepithelium. Other CNS regions from whichNSCs can be isolated include the ventricular and subventricular zones ofthe CNS and other CNS regions which include mitotic precursors as wellas post-mitotic neurons. In an embodiment, the disclosed methods canemploy NSCs residing in regions of a developing mammalian CNS.

In an embodiment, the NSCs are obtained from an area which is naturallyneurogenic for a desired population of neurons. The desired populationof cells may include the cells of a specific neuronal phenotype whichcan replace or supplement such phenotype lost or inactive in aneurological condition.

A variety of different neuronal subtypes, including those useful fortreatment of specific neurodegenerative diseases or conditions can beobtained by isolating NSCs from different areas or regions of the CNSand across different gestational ages during fetal development. NSCsisolated from different areas or regions of the CNS and across differentgestational ages are used for optimal expansion and neuronaldifferentiation capacity. One of the hallmarks of the mammalian CNS isthe diversity of neuronal subtypes. A single population of NSCs, forexample, may spontaneously generate only a few distinct neuronalsubtypes in culture. Furthermore, the cells from a particular fetalgestational age may establish the physiological relevance of thecultured cells.

In an embodiment of the disclosed methods, the cells to be transplantedinto subjects are derived from the human fetal counterpart of theinjured neural area. In an embodiment, NSCs are isolated from humanfetal CNS regions at gestational ages of between about 6.5 to about 20weeks. In an embodiment, cells from a fetal spinal cord are isolated ata gestational age of about 7 to about 9 weeks. It should be appreciatedthat the proportion of the isolatable neural stem cell population canvary with the age of the donor. Expansion capacity of the cellpopulations can also vary with the age of the donor. Such regional andtemporal specificity of NSCs indicates that NSCs behave asfate-restricted progenitors and not as blank cells or a singlepopulation of cells.

The proportion of the population in vitro including GABA-producingneurons is generally constant at about 5-10%.

The NSCs of the ventral midbrain, for example, are distinct from theNSCs obtained from the spinal cord at the same gestational stage. Inparticular, the NSCs from the ventral midbrain exclusively give rise totyrosine-hydroxylase-expressing dopaminergic neurons, whereas NSCs fromthe spinal cord exclusively generate acetylcholine-producing cholinergicneurons. Both cell types, however, simultaneously generate the moreubiquitous gluamate- and GABA-producing neurons. Therefore, in anembodiment, the disclosed methods include obtaining NSCs from theventral midbrain to treat conditions ameliorated or attenuated, at leastin part, by the implantation of tyrosine-hydroxylase-expressingdopaminergic neurons. The disclosed methods further include obtainingNSCs from the spinal cord to treat neurodegenerative conditionsameliorated or attenuated, at least in part, by the implantation ofacetylcholine-producing cholinergic neurons.

Thus, for treatment of movement disorders such as Parkinson's diseasewhich is characterized by the loss of dopaminergic neurons, anembodiment of the disclosed methods includes the use of NSCs derivedfrom an area such as the ventral midbrain in which neurogenesis ofdopaminergic neurons is substantial. In addition, the NSCs can beobtained at a gestational age of human fetal development during whichneurogenesis of dopaminergic neurons is substantial. Accordingly, in anembodiment, the disclosed methods include obtaining NSCs from theventral midbrain derived at a gestational age of about 7 to about 9weeks to treat movement disorders.

For treating motor neuron diseases such as amyotrophic lateral sclerosisor flaccid paraplegia resulting from loss of ventral motor-neurons, anembodiment of the disclosed methods includes the use of NSCs derivedfrom an area such as the spinal cord in which neurogenesis of ventralmotor-neurons is substantial and obtained at a gestational age of humanfetal development during which neurogenesis of ventral motor-neurons issubstantial. Accordingly, in an embodiment, NSCs are isolated from thespinal cord at a gestational age of about 7 to about 9 weeks to treatmotor neuron diseases.

It should be appreciated, however, that, in some cases, the limits ofsuch regional specificity are fairly broad for practical purposes. Thus,NSCs from various areas of the spinal cord such as cervical, thoracic,lumbar, and sacral segments can be used interchangeably to be implantedand to treat locations other than the corresponding origin of the NSCs.For example, NSCs derived from cervical spinal cord can be used to treatspasticity and/or rigidity by transplanting the cells into the lumbarsegments of a patient.

NSCs can also be isolated from post-natal and adult tissues. NSCsderived from post-natal and adult tissues are quantitatively equivalentwith respect to their capacity to differentiate into neurons and glia,as well as in their growth and differentiation characteristics. However,the efficiency of in vitro isolation of NSCs from various post-natal andadult CNS can be much lower than isolation of NSCs from fetal tissueswhich harbor a more abundant population of NSCs. Nevertheless, as withfetal-derived NSCs, the disclosed methods enable at least about 30% ofNSCs derived from neonatal and adult sources to differentiate intoneurons in vitro. Thus, post-natal and adult tissues can be used asdescribed above in the case of fetal-derived NSCs, but the use of fetaltissues is preferred.

Various neuronal subtypes can be obtained from manipulation of embryonicstem cells expanded in culture. Thus, specific neuronal subtypes, basedon the disclosed methods, can be isolated and purified from otherirrelevant or unwanted cells to improve the result, as needed, and canbe used for treatment of the same neurodegenerative conditions.

The NSCs in the disclosed methods can be derived from one site andtransplanted to another site within the same subject as an autograft.Furthermore, the NSCs in the disclosed methods can be derived from agenetically identical donor and transplanted as an isograft. Stillfurther, the NSCs in the disclosed methods can be derived from agenetically non-identical member of the same species and transplanted asan allograft. Alternatively, NSCs can be derived from non-human originand transplanted as a xenograft. With the development of powerfulimmunosuppressants, allograft and xenograft of non-human neuralprecursors, such as neural precursors of porcine origin, can be graftedinto human subjects.

A sample tissue can be dissociated by any standard method. In anembodiment, tissue is dissociated by gentle mechanical trituration usinga pipet and a divalent cation-free saline buffer to form a suspension ofdissociated cells. Sufficient dissociation to obtain largely singlecells is desired to avoid excessive local cell density.

For successful commercial application of NSCs, maintaining robust andconsistent cultures that have stable expansion and differentiationcapacities through many successive passages is desirable. As describedabove, the culture methods can be optimized to achieve long-term, stableexpansion of an individual cell line of NSCs from different areas andages of CNS development while maintaining their distinct progenitorproperties.

To this end, it has been surprisingly found that promoting the adhesionof NSCs (NSCs) to a substrate contributes to accelerating the mitoticrate of NSC or progenitor cells, thereby, providing significantenhancement of a more robust culture of NSC or progenitor cells. Inparticular, in addition to avoiding excessive local cell density andmaintaining mitogen concentrations, it has been found that theconcentrations of extracellular matrix proteins affect the long-termmitotic and differentiation capacity of NSCs. The extracellular matrixproteins can include poly-D-lysine, poly-L-lysine, poly-D-ornithine,poly-L-ornithine, fibronectin and combinations thereof. Otherextracellular matrix proteins can include various isotypes, fragments,recombinant forms, or synthetic mimetics of fibronectin, lamin,collagen, and their combinations. Alternatively, or in addition, itshould be appreciated that the disclosed methods can include any othersuitable substance that is able to promote effective cell adhesion sothat each individual cell is adhered to the culture substrate during theentire duration of the culture without being cytotoxic or retarding thecell division.

Although extracellular matrix proteins can be effective in promotingcell adhesion, different amino acid polymers, such as poly-L/D-ornithineor poly-L/D-lysine, can be toxic to the cells at certain concentrationsfor each individual cell line. The duration of incubation can alsoaffect the final amount of the polymer deposited on the dish surfaceaffecting the viability of the cells. For the NSCs employed in thedisclosed methods, concentrations of polymer can be within a range ofbetween about 0.1 μg/mL and about 1 mg/mL. In an embodiment, 100 μg/mlof poly-D-lysine is dissolved in 0.01M HEPES buffer or water at neutralpH and applied to a culture vessel. The culture vessel is incubated for1 hour at room temperature. The culture vessel is then thoroughly rinsedwith water and dried prior to use.

The disclosed methods can also include double-coating the culturevessels with an extracellular matirx protein. In an embodiment, theculture vessel is treated with fibronectin or a fibronectin derivativefollowing the application of poly-L/D-ornithine or poly-L/D-lysinedescribed above. In an embodiment, fibronectin protein prepared fromhuman plasma is used. It should be appreciated, however, that any othersuitable form or source of fibronectin protein can be used such asporcine or bovine fibronectin, recombinant fibronectin, fragments offibronectin proteins, synthetic peptides, and other chemical mimetics offibronectin. In an embodiment, between about 0.1 μg/mL to about 1 mg/mLof fibronectin can be applied.

In an embodiment including the expansion of NSCs from human spinal cord,the culture vessel is treated with 100 μg/mL of poly-D-lysine for aperiod sufficient to allow the extracellular protein to bind to and coatthe culture vessel. Such a time period can be for about five minutes toabout three hours. The culture vessel can be subsequently washed withwater. After air-drying the culture vessel, the vessel can be treatedwith about 25 mg/ml fibronectin for approximately five minutes toseveral hours at room temperature or about 1 mg/ml fibronectin forapproximately 1 hour to several days at 37° C. Subsequently, thefibronectin can be removed and the culture vessel can be washed at leastonce or stored in PBS until use.

Alternatively, fibronectin can be added into the growth medium as asoluble factor supplied directly to the cells. In this embodiment, NSCscan be expanded by adding 1 μg/mL of fibronectin into the growth mediumin addition to, or instead of, treatment of the culture vessels withfibronectin. Supplying the attachment protein into the growth medium asa soluble factor at the time of cell plating is particularlyadvantageous for large commercial-scale culturing of NSCs due to therelatively short shelf-life of fibronectin-coated vessels. This methodis also useful for manufacturing a neural stem cell line requiringsubstantially exact conditions and reproducibility such as requiredunder cGMP protocols and for manufacturing the neural stem cell line fortherapeutic use.

In an embodiment, the isolated NSCs are added to the culture vessel at adensity of about 1,000 to about 20,000 cells per square cm. Such adensity contributes to even dispersion and adhesion of individual cellsin the culture vessel, avoiding localized concentrations of cells, toenriched the culture for NSCs.

In an embodiment, NSCs are expanded in the absence of serum. In anembodiment, the NSCs are cultured in a defined, serum-free medium toavoid exposure of the NSCs to concentrations of serum sufficient todestabilize the mitotic and differentiation capacity of NSC. Inaddition, exposure of the NSCs to certain growth factors such asleukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF)can also destabilize NSCs and should be avoided.

Mitogens can be added to the culture at any stage of the culture processto enhance the growth of the NSCs. Mitogens can include basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermalgrowth factor (EGF), transforming growth factor-alpha (TGFa), andcombinations thereof.

NSCs of the disclosed methods can be grown and expanded in at least twodifferent culture forms. One form of culture includes an aggregatedform, commonly referred to as a clustered, aggregated form referred toas a suspension culture. Another form of culture includes a dispersed,non-aggregated form referred to as an adhesion culture.

In a dispersed adherent culture of the NSCs of the disclosed methods,the cells form a monolayer in which individual cells initially contactthe culture substrate directly. Eventually, after a period ofincubation, the cells can sporadically form clusters, wherein at leastone additional layer of cells is formed on the bottom layer, even as thecells in the bottom layer are individually adhered to the substrate.Such clustering especially occurs when the culture is inoculated at highcell density or allowed to reach high cell density, which, in anembodiment, is minimized for optimal expansion of NSCs or progenitorcells or for optimal maintenance of the multipotential capacity of theNSCs. In the dispersed adherent culture of an embodiment of thedisclosed methods, human NSCs are enabled to divide in less than aboutfour days per cell division.

Another distinctive characteristic of the dispersed adherent culture isthat the NSCs of the disclosed methods divide to generate daughtercells, each retaining their multipotential capacity. In an embodiment,the dispersed adherent culture of NSCs of the disclosed methods includesan expansion capacity of at least 20 cell doublings in the absence ofsubstantial differentiation. Most NSCs can be expanded beyond at least50 cell doublings before losing their neurogenic potential. In anembodiment, the NSCs expanded in the dispersed adherent culture of thedisclosed methods demonstrate enhanced neuronal differentiation, givingrise, in an embodiment, to at least about 30% neuronal differentiation.In many cases, at least 50% of NSCs differentiate into neurons. Althoughthe dispersed, adherent form of culture is a more preferred form ofculture, the different culture methods may allow isolation of innatelydistinct cell populations with differing differentiation potentialseither in vitro or in vivo.

The present method also allows clonal isolation of NSCs from a varietyof sources without genetic modification or inclusion of feeder cells.Thus, a very low number, preferably less than 1000 cells per squarecentimeter of cells, may be seeded in a cell culture dish prepared asdescribed above.

A few days following seeding of the NSCs, the cells can formwell-isolated colonies. The colonies may be grown to a desired size suchas at least about 250 to about 2000 cells. In an embodiment, at leastone colony of cells is manually picked and inoculated individually to afresh cell culture dish such as a multi-well plate.

Isolated clonal populations may be expanded by serial passaging and usedto establish multiple neural stem cell lines. Many such clonal celllines have been isolated from various areas of the human CNS includingspinal cord, midbrain, and hindbrain. Clonal cell lines are useful toenrich for a particular cell phenotype such as a higher proportion ofneuronal subtypes. For example, clonal cell lines enriched for tyrosinehydroxylase-expressing dopaminergic neurons, GABAergic neurons,cholinergic neurons and neurons of other specific phenotypes can beisolated with the disclosed methods.

In an embodiment, either a polyclonal or a monoclonal neural stem cellline can be induced to be further enriched for a particular subtype ofneurons. A number of growth factors, chemicals, and natural substanceshave been screened to identify effective inducers of particular neuronssuch as tyrosine hydroxylase-expressing dopaminergic neurons andacetylcholine-producing cholinergic neurons from NSCs of midbrain orspinal cord. The factor or chemical or combination thereof can beintroduced during the mitotic phase and/or the differentiation phase ofthe NSCs. In an embodiment, a neural stem cell line for a dopaminergicphenotype is further enriched as a donor population to treat Parkinson'sdisease.

Various neuronal subtypes can be obtained from isolation of stem cellshaving a desired differentiation pattern in vitro. In vitro results canbe substantially reproduced in vivo. This means that the potentialefficacy of the stem cells in vivo can be predicted by thedifferentiation pattern of the stem cells in vitro. Upon injection intolive post-natal subjects, NSCs, in either an undifferentiated orpre-differentiated state, produces to a large extent an in vivodifferentiation pattern observed in vitro. Thus, NSC giving rise totyrosine hydroxylase-producing neurons in vitro also generate tyrosinehydroxylase-producing neurons in vivo. Conversely, NSCs not giving riseto tyrosine hydroxylase-producing neurons constitutively in vitro do notproduce tyrosine hydroxylase-producing neurons in vivo.

However, differentiation cues present in vitro are limited compared tothose in vivo. Thus, a substantial fraction of the differentiatedneurons may not express a major neurotransmitter phenotype. Additionalcues such as signals from afferent or efferent neurons, or agentsmimicing such natural signals can be used to re-configure thedifferentiated phenotypes, either during the mitotic stage of NSCs orduring their differentiation. NSCs have the ability to respond to cuespresent in vivo as well as in vitro. Thus, once grafted intoischemia-injured spinal cord, the spinal NSCs generate a substantiallyhigher proportion of GABA-producing neurons than in vitro. Thus, NSCsare plastic. Such plastic nature of NSCs is a characteristic of theirmultipotentiality and, as such, this plasticity can be used to identifyphenotype-inducing agents and conditions which can be further combinedwith an NSC population to re-direct its properties.

In an embodiment, such re-programming includes treating NSCs from aspinal cord tissue to obtain enhanced expression of motor neuronphenotypes. The treatment conditions include co-culturing NSCs or theirdifferentiated cells with various muscle cells or peripheral nervoussystem-derived cells such as neural crest cells or ganglionic neurons.NSCs can also be treated with cocktails of molecules known to beexpressed and produced in motor neurons or in the spinal cord to enhanceNSC expression of motor neuron phenotypes.

To induce enhanced NSC expression of the dopaminergic phenotype of humanmidbrain, NSCs are treated with molecules such as lithium, GDNF, BDNF,pleiotrophin, erythropoeitin, conditioned media from cells such assertoli cells, or any other suitable chemicals or cells obtained byscreening or combinations thereof. Such inducement can enable thetransplanted NSCs to express and maintain the dopaminergic phenotype invivo.

In an embodiment, the NSCs of the disclosed methods can includepre-differentiated cells for transplantation. For maximum yield of thecells and for simplicity of the procedure, a confluent culture isharvested for transplantation which comprises primarily a population ofundifferentiated cells. It should be appreciated, however, that a minorpopulation of cells just starting to differentiate spontaneously canalso exist due to the increased cell density.

In an embodiment, passaging NSCs includes harvesting or detaching thecells from a stubstrate. In an embodiment, the disclosed methods includeharvesting or detaching the cells from a stubstrate using at least oneenzyme. Enzymatic treatment can be avoided when the cell cycle time ofthe NSCs is short enough to deactivate the mitogen receptors on the cellsurface. However, the cell cycle time of human NSCs is much longer thanrodent NSCs such that human NSCs are not as sensitive to enzymatictreatment. Thus, in the disclosed methods, enzymatic treatment is usedfor harvesting NSCs derived from a human. Although the human NSCs canbecome temporarily refractory to mitogen in the presence of enzymatictreatment, repeated deactivation of the mitogen receptors can lead to adecreased proportion of NSCs.

In an embodiment, upon harvesting of the cells, the cells areconcentrated by brief centrifugation. The cells can be further washedand re-suspended in a final, clinically usable solution such as saline,buffered saline, or, alternatively, be re-suspended in a storage orhibernation solution. Alternatively, the cells can be re-suspended in afreezing medium such as media plus dimethylsulfoxide, or any othersuitable cryoprotectant, and frozen for storage.

The hibernation solution is formulated to maintain the viability of livecells for a prolonged period of time. In an embodiment, the storagesolution can be adapted to be used for shipping live cells in aready-to-use formulation to a transplantation surgery site for immediateuse. Suitable conditions for shipping live cells to a distant site alsoincludes an insulation device that can maintain a stable temperaturerange between about 0° C. and about 20° C. for at least 24 hours. Livecells stored at between about 0° C. and about 8° C. for about 24 hoursto about 48 hours are engraftable for treatment of a disease orcondition.

In an embodiment, the cells are concentrated in a solution such as theclinically usable, hibernation or freezing solutions described above. Inan embodiment, the cells are concentrated to an appropriate cell densitywhich can be the same or different from the cell density foradministration of the cells. In an embodiment, the cell density foradministration can vary from about 1,000 cells per microliter to about1,000,000 cells per microliter depending upon factors such as the siteof the injection, the neurodegenerative status of the injection site,the minimum dose necessary for a beneficial effect, and toxicityside-effect considerations. In an embodiment, the disclosed methodsinclude injecting cells at a cell density of about 5,000 to about 50,000cells per microliter.

The volume of media in which the expanded cells are suspended fordelivery to a treatment area can be referred to herein as the injectionvolume. The injection volume depends upon the injection site and thedegenerative state of the tissue. More specifically, the lower limit ofthe injection volume can be determined by practical liquid handling ofviscous suspensions of high cell density as well as the tendency of thecells to cluster. The upper limit of the injection volume can bedetermined by limits of compression force exerted by the injectionvolume that are necessary to avoid injuring the host tissue, as well asthe practical surgery time.

Low cell survival of donor cells using known methods has necessitatedthe delivery of a large quantity of cells to a relatively small area inorder to attempt effective treatment. Injection volume, however, ishydrostatic pressure exerted on the host tissue and the prolongedinjection time associated with high injection volumes exacerbatessurgical risk. Additionally, over-injection of donor cells leads tocompression and subsequent injury of the host parenchymal tissue. Inattempting to compensate for volume constraints, known methods haverequired preparation of high cell density suspensions for theinjections. However, a high cell density promotes tight clustering ofthe transplanted cells and inhibits cell migration or spreadingpreventing effective treatment beyond a limited area and compromisingseamless integration into the host tissue.

In contrast, as a result of improved survival in vivo of the cellsprepared by the disclosed methods, fewer number of cells are needed perinjection. In fact, up to three to four times the number of injectedcells have been shown to exist after six months from the time ofinjection demonstrating significant quantitative survival using thedisclosed methods. Also, because of the quantitative survival,reproducible administration of desired cell doses can be achieved.Accordingly, in an embodiment, the cells are concentrated to a densityof about 1,000 to about 200,000 cells per microliter. In an embodiment,about 5,000 to about 50,000 cells per microliter have been used foreffective engraftment. In another embodiment, about 10,000 to 30,000cells per microliter is used. In an embodiment, the cells can bedelivered to a treatment area suspended in an injection volume of lessthan about 100 microliters per injection site. For example, in thetreatment of neurodegenerative conditions of a human subject wheremultiple injections may be made bilaterally along the spinal tract, aninjection volume of 0.1 and about 100 microliters per injection site canbe used.

Any suitable device for injecting the cells into a desired area can beemployed in the disclosed methods. In an embodiment, a syringe capableof delivering sub-microliter volumes over a time period at asubstantially constant flow rate is used. The cells can be loaded intothe device through a needle or a flexible tubing or any other suitabletransfer device.

In an embodiment, the desired injection site for treatment of aneurodegenerative condition includes at least one area of the spinalcord. In an embodiment, the cells are implanted into at least onespecific segment or region of the spinal cord such as the cervical,thoracic or lumbar region of the spinal cord. In the lumbar region, forexample, only five pairs of nerve roots traverse the bony canal ofvertebrae with each pair of nerve roots exiting the spine at each lumbarlevel distributed over a wide area. Due to a lower density of nerveroots in the lumbar region of the spinal cord, the lumbar region isparticularly well-suited for providing a safe site for injection ofcells. In an embodiment, the cells are implanted in the intermediatezone of the spinal cord parenchyma.

In an embodiment, the cells are injected at between about 5 and about 50sites. In an embodiment, the cells are injected at between about 10 toabout 30 sites on each side of the cord. At least two of the sites canbe separated by a distance of approximately 100 microns to about 5000microns. In an embodiment, the distance between injection sites is about400 to about 600 microns. The distance between injections sites can bedetermined based on generating substantially uninterrupted andcontiguous donor cell presence throughout the spinal segments and basedon the average volume of injections demonstrated to achieve about 2-3month survival in animal models such as rats or pigs. In an embodiment,the cells are injected along both sides of the midline of the spinalcord to span the length of at least several lumbar segments useful fortreating a symptom such as spasticity/rigidity or motor neuron survival.The actual number of injections in humans can be extrapolated fromresults in animal models.

In an embodiment, the target site of injection is the gray matter of thespinal cord. Within the gray matter, the needle tip can be positioned todeposit the NSCs at specific levels of lamina. For instance, to deliverGABA/glycine-producing neurons to treat spasticity/rigidity, the NSCsare delivered to the area encompassing lamina V-VII. Alternatively, theNSCs can be delivered to or near the dorsal horn of the gray matter ofvarious spinal segments, from cervical to lumbar, in order to treatneuropathic pain or chronic pain. Alternatively, the NSCs can bedelivered to or near the ventral horn of the gray matter of variousspinal segments from cervical to lumbar in order to treat motor neurondiseases such as ALS.

The cells of the disclosed methods can generate large numbers of neuronsin vivo. When the NSCs are not overtly pre-differentiated prior totransplant, the NSCs can proliferate up to two to four cell divisions invivo before differentiating, thereby further increasing the number ofeffective donor cells. Upon differentiation, the neurons secretespecific neurotransmitters. In addition, the neurons secrete into themileu surrounding the transplant in vivo growth factors, enzymes andother proteins or substances which are beneficial for differentconditions. Accordingly, a variety of conditions can be treated by thedisclosed methods because of the ability of the implanted cells togenerate large numbers of neurons in vivo and because theneurodegenerative conditions may be caused by or result in missingelements including neuron-derived elements. Therefore, subjectssuffering from degeneration of CNS tissues due to lack of suchneuron-derived elements, such as growth factors, enzymes and otherproteins, can be treated effectively by the disclosed methods.

Conditions responding to growth factors, enzymes and other proteins orsubstances secreted by the implanted neurons include hereditarylysosomal diseases such as Tay-Sach's disease, Niemann-Pick's disease,Batten's disease, Crabb's disease, ataxia, and others.

In addition, the disclosed methods of treatment include implanting thecells expanded in vitro which can replace damaged or degeneratedneurons, provide an inhibitory or stimulatory effect on other neurons,and/or release trophic factors which contribute to the regeneration ofneurons.

An embodiment includes supplying additional motor neurons as replacementof damaged or degenerated neurons. For example, the disclosed methodsinclude providing sufficient neural infrastructure within the syrinx ofthe spinal cord to fill the cavitation. Neural infrastructure issufficient if it is capable of slowing the enlargement of the syrinxassociated with syringomyelia resulting from traumatic spinal injury,hereditary conditions or any other cause. It should be appreciated thatproviding sufficient neural infrastructure also helps relieve furthercomplications arising from degenerating spinal cord.

Not all NSCs are therapeutic for a given disease. The types of neuronalpopulations affected in different diseases may be different. Therefore,a therapeutically effective donor population of NSCs contributes toreplacing the lost neural element. For example, treatment of spasticity,seizure, movement disorders, and other muscular hyperactivity disorders,can include providing a therapeutically effective amount of cellscapable of differentiating into inhibitory neurons producing GABA orglycine. Distinct populations of NSCs can be assessed in vitro byexamining the differentiated neuronal phenotype. The in vitrodifferentiation pattern is then used to predict the efficacy of thecells to produce the appropriate phenotype in vivo, not only in terms ofan appropriate neurotransmitter phentoype, but also in terms of anappropriate morphology, migration, and other phenotypic characteristicsof neurons.

In an embodiment, NSCs are implanted that are capable of generating theneuronal subtype corresponding to the damaged or destroyed neuronalsubtypes associated with the etiology of the symptoms. For example,hyperactivity of excitatory circuits in subjects can be caused byhereditary conditions or neuronal injury from spinal trauma,thoracic/thoracoabdominal aorta surgery, stroke, epilepsy, brain trauma,Huntington's disease, bladder incontinence, hyperactive bowel movementand any other uncontrolled contraction of muscles arising from injury orhereditary conditions. Spasticity, seizures, or other hyperactivityoccurs in the brain as opposed to the spinal cord due to a number ofdifferent etiological origins. Focal epilepsy, for example, is thoughtto arise from disregulated hyperactivity due to a lack of GABA exertingtonal control over the circuitry. To this end, disclosed methods includeproviding inhibitory neurotransmitters such as GABA or glycine in theaffected areas by transplantation of in vitro expanded NSCs. In the caseof spasticity, seizure, and other hyperactivity, for example, a numberof NSCs which are capable of differentiating into inhibitory neurons,such as GABA-producing or glycine-producing neurons, are generated invivo to be transplanted to attenuate at least one hyperactive neuralcircuit associated with spasticity, seizure, and other neuronalhyperactivity. The disclosed methods can, therefore, be applied to treatepilepsy and similar conditions of seizures.

The disclosed methods can also be applied to treat paresis, paralysis,spasticity, rigidity or any other motor, speech, or cognitive symptomsarising from cerebral ischemia. Cerebral ischemia can occur as a resultof a stroke event in the brain or from a heart attack in which the bloodcirculation to the brain is interrupted for a significant period oftime. It is, thus, analogous to the spinal cord ischemia describedabove. Some stroke subjects develop seizures of central origin as wellas other deficits such as memory loss, paralysis, or paresis. Thesedeficits from cerebral ischemia are also likely due to selective loss ofinhibitory interneurons in hippocampus and/or other brain areas. Thus,the disclosed methods can be applied to treat stroke subjects sufferingfrom paresis, paralysis, spasticity, or other motor, speech, andcognitive symptoms.

In the case of paresis, flaccid paraplegia, and other conditionsassociated with a loss of control of muscle contraction such as thosecaused by ALS, traumatic spinal cord injury, ischemic injury, orhereditary conditions, the disclosed methods include providing neuronalimplantation to exert sufficient trophic influence to slow the loss ofmotor neurons. In particular, the disclosed methods facilitate theability of the transplanted NSCs to secrete trophic molecules which canbe delivered to degenerating motor neurons under conditions of optimalbiovailability. Such trophic molecules include exocitosed superoxidedismutases such as superoxide dismutase (SOD1), lysosomal enzymes, andnon-proteinatious molecules such as cell-produced antioxidants. Othertrophic factors secreted by the transplanted cells can include globalcell-derived neurotrophic factor (GDNF), brain-derived neurotrophicfactor (BDNF), vascular epidermal growth factor (VEGF), pleiotrophin,vascular endothelial growth factor (VEGF), erythropoetin, midkine,insulin, insulin-like growth factor 1 (IGF-1), and insulin-like growthfactor 2 (IGF-2) or any other beneficial trophic element.

Another factor that contributes to the ability of the disclosed methodsto treat a wide range of neurodegenerative conditions includes theability of the NSC-differentiated cells to migrate extensively alongexisting neuronal fibers. Migration of the grafted cells results inglobal distribution and integration of donor neurons and/or glia andglobal or dispersed supply of the therapeutic element secreted by suchcells.

Wide migration of the cells enables the global and stable delivery ofkey therapeutic proteins and substances throughout a nervous system andbody of a subject in need thereof. Thus, the cells of the disclosedmethods are effective delivery vehicles for therapeutic proteins andsubstances. For such delivery purposes, the disclosed methods includetransplanting the cells into various sites within the nervous systemincluding the CNS parenchyma, ventricles, the subdural, intrathecal andepidural spaces, peripheral nervous system sites as well as into areasoutside of the nervous system including the gut, muscle, endovascularsystem, and subcutaneous sites.

EXAMPLE 1 Expansion of Human Spinal Cord Neural Stem/Progenitor Cells

Spinal cord from at least one donor of gestational age of approximately7-8.5 weeks is obtained. A single contiguous tissue of the spinal cordis dissociated in Ca⁺⁺ and Mg⁺⁺-free phosphate buffered saline usingmechanical trituration. The resulting cell suspension is then seededinto tissue culture plates pre-coated with both poly-L-ornithine orpoly-D-lysine and human fibronectin or other extracellular matrixproteins. Tissue culture-treated plates or flasks were incubated with100 μg/ml poly-D-lysine for 1 hour at room temperature. They were thenwashed three times with water and dried. They were then incubated with25 mg/ml for 5 minutes at room temperature. Sometimes, 10 mg/mlfibronectin for 1 hour at room temperature was used. Sometimes, 1 mg/mlfibronectin for 18 hours at 37° C. was used. Culture media consisting ofN2 (DMEM/F12 plus insulin, transferrin, selenium, putrescine, andprogesterone) was supplemented with 1 human recombinant basic fibroblastgrowth factor (bFGF). In an embodiment, a range of 0.1 ng/ml-100 ng/mlcan be used. In an embodiment, optimally, 10 ng/ml of bFGF is used.

The resulting initial culture consists of post-mitotic neurons andproliferative NSCs in a monolayer. Subsequently, after approximatelyfive to about twenty days in culture, the dividing, nestin-positive,NSCs dominate the culture over the non-dividing neurons or theslowly-dividing glia. Under these culture conditions, NSCs areselectively favored for expansion. The expanding NSC population ispassaged by mild enzymatic treatment, such as using trypsin. The cellsare cultured in media free of serum or substantially free of serum.Although low concentration of serum may be tolerated by the cells, it isbest to avoid exposing the cells to serum since serum contains manycytokines such as LIF and CNTF which promote glial differentiation ofthe NSCs. Thus, during passage, the enzyme used is stopped by addingspecific enzyme inhibitor, such as trypsin inhibitor, rather than serum.At each passage, the number of harvested cells are counted, and afraction is re-seeded for further expansion. As illustrated in FIG. 1,using the method of the instant invention, human NSCs can be expandedbeyond 10¹⁸-fold increase in population while maintaining their growthand differentiation properties. The cells can be expanded reproducibly.As shown in FIG. 1, serial passaging of the cells have been repeatedthree times with reproducible growth curve and doubling time of thecells. During the expansion, almost all cells express nestin, the invivo marker of mitotic neuroepithelial cells, and are absent of antigensof differentiated neurons and glia such as type 3-beta tubulin and GFAP.The cells are also negative by immunostaining for PSA-NCAM, a possiblemarker of committed neuronal progenitors, O4 and GalC, markers ofoligodendrocytes, and RC2, a marker of radial glia. Thus, determined byimmunostaining, the NSCs stably maintain their expression of antigenprofile throughout the prolonged expansion period. An example of themorphology and the nestin expression is shown in FIGS. 2A and B,respectively.

EXAMPLE 2 Differentiation of Human Spinal Cord Neural Stem/ProgenitorCells

At any point during expansion of the NSCs, the cultures can bedifferentiated by withdrawal of the mitogen in the culture such as bFGF.Differentiation of NSCs ensues within about 1-3 days after the removalof mitogen, and distinct heterogeneous cell morphologies are apparent.By approximately day 4-7 of differentiation, neuron-specific antigens,such as MAP2c, tau, and type III beta-tubulin, can be visualized byimmunostaining. By approximately day 12-14, elongated, fasciculatedaxonal processes are evident throughout the culture along with clearpolarization of subcellular protein trafficking. By approximately day28, synaptic proteins, such as synapsin and synaptophysin, localize intoaxon terminals, appearing as punctate staining. Additional feeder layerof astrocytes can be provided to further promote long-term maturation ofthe neurons. As illustrated in FIG. 3, differentiation of human spinalNSCs generates mixed cultures of neurons and glia wherein the neuronsrobustly express neuron-specific antigens such as tau, MAP2ab (A) andtype3 beta tubulin (B) and comprises approximately 50% of the culture.As shown in FIG. 3B, the culture spontaneously generates long, bundled,axon cables that stretch for several centimeters. As illustrated in FIG.3C, significant proportion of the neurons are GABAergic. Cholinergicmotor neurons are also present in the culture (FIG. 3D). Presence ofsignificant GABA neurons in culture predicts usefulness of the humanspinal NSCs for treating various neurological conditions caused bydecreased GABA production in certain circuitry. Likewise, presence ofcholinergic neurons demonstrates that the human spinal NSCs are capableof motor neuron differentiation and predicts their usefulness fortreating various motor neuron diseases caused by gradual degeneration ofmotor neurons. For treatment, the NSCs are expanded with or withoutfurther phenotype-enhancing conditions, harvested, and injected into aneural area of deficiency.

EXAMPLE 3 Expansion of Human Midbrain Neural Stem/Progenitor Cells

One midbrain tissue of fetal gestational age 7-8.5 week is obtained.NSCs from the midbrain tissue are obtained as described in Example 1.The cells are serially passaged over 160 days of net culture period andthe resultant expansion is shown in FIG. 4. Throughout the period of theexpansion, the NSCs stably maintain their multipotipotentiality andneurogenic potential as well as their differentiation potential to giverise to dopaminergic neurons. Dopaminergic neurons are assessed byneuronal expression of tyrosine hydroxylase (TH) and dopaminetransporter (DAT).

DAT expression is a marker of dopamine-producing neurons. DAT expressionin neurons can be assessed by measuring its function to transportradiolabelled dopamine across the synaptic membrane of differentiatedneurons in culture. DAT function in cultures from differentiated humanmidbrain NSCs and monoclonally derived human midbrain NSCs are assessedby the radiolabelled dopamine uptake assay (FIG. 5). The assay resultshows robust functional dopaminergic activity of human midbrain NSCs.Moreover, it illustrates that dopaminergic phenotype can be enriched byisolated monoclonal population of NSC which is particularly inclined togive rise to higher proportion of dopaminergic neurons upondifferentiation (FIG. 5).

NSCs generating enriched dopaminergic neurons is particularly useful fortreatment of parkinson's disease. Similarily, NSCs pre-programmed at thetime of isolation from the tissue for enhanced differentiation for aspecific phenotype can be used to isolate other specific desiredneurons, such as forebrain cholinergic neurons useful for treatment ofAlzheimer's disease, spinal cholinergic neurons useful for treatment ofmotor neuron diseases such as ALS, serotonergic neurons useful fortreatment of depression, and GABAergic neurons useful for treatment ofepilepsy and Hungtinton's disease.

EXAMPLE 4 Differentiation of Human Midbrain Neural Stem/Progenitor Cells

Human midbrain NSCs/progenitors can be differentiated as described inExample 2. During the mitotic period of the NSCs or during theirdifferentiation, the proportion of desired phenotype can be enriched bytreatment of the culture with various exogenous factors. An example ofsuch factors capable of enriching dopaminergic phenotype from the humanmidbrain NSCs is demonstrated by the conditioned media from sertolicells, as illustrated FIGS. 6A and 6B.

In the studies shown in in FIGS. 6A and 6B, cryopreserved neural stemcells were thawed and plated at a density of 40,000 cells per well in4-well chamber slides in the presence of mitogen and allowed toproliferate for 6 days, at which time mitogen was removed and cells wereallowed to differentiate for 8 days. Cells were divided into four groupsbased on the timing and duration of exposure to sertoli cell conditionedmedium (SCCM, diluted 1:1 in N2a). One group was exposed to SCCM duringproliferation and differentiation (condition 1); a second was exposedduring proliferation only (condition 2); a third was exposed duringdifferentiation only (condition 3); and a fourth was not exposed to SCCM(control or cont.). Media was changed every other day, and mitogen wasadded daily during the proliferative phase. Four wells were maintainedper condition to allow staining for multiple markers, and three celllines were tested: 796MB, 527MB, and 566SC. 566SC cells, which werederived from spinal cord did not have measurable TH-positive neurons,were omitted from the figure.

Upon differentiation cells were fixed using 4% paraformaldehyde andimmunostained using antibodies to MAP2 [AP20 clone (Sigma) whichrecognizes MAP2ab subtypes], neuron-specific β-tubulin [TuJ1 (Covance)],and tyrosine hydroxylase (Pel-Freez), as well as GFAP (Dako) and GalC(Chemicon). Immunostained cells were counted using a 40× objective, andat least three fields were counted for each well. Few or no GFAP+ orGalC+ cells were detected upon analysis of cells maintained under anycondition, so these antigens were excluded from the analysis. Inaddition, 566SC cells were too dense to quantitate after maintenanceunder the conditions described and were not included in the finalanalysis.

The analysis illustrates that the human midbrain NSCs can be influencedby treatment with exogenous factors to search for useful protein factoror chemicals to further enrich for dopaminergic neurons. Thus, novelsynthetic/natural chemical and protein factors can be effectivelyscreened by using human NSCs to obtain particularly useful populationsfor treatment of specific indication such as Parkinson's disease.

EXAMPLE 5 Treatment of Spasticity and Rigidity in Rats byTransplantation of Human Spinal Neural Stem/Progenitor Cells

To induce transient spinal cord ischemia the technique previouslydescribed in Taira, (1996) is used. Sprague Dawley (SD) rats areanesthetized in halothane (1.5%). A 2 Fr Fogarty® catheter is passedthrough the left femoral artery and through the descending thoracicaorta to the level of the left subclavian artery. To measure distalarterial pressure (DBP) below the level of aortic occlusion, the tailartery is cannulated with a polyethylene (PE-50) catheter.

Spinal cord ischemia is induced upon inflation of the intra-aorticballoon catheter with 0.05 mL of saline. Systemic hypotension during theperiod of aortic occlusion is reproduced by withdrawing a portion ofarterial blood (10.5-11 cc) from a carotid artery cannulated with aPE-50 catheter. Systemic hypotension of approximately 40 mm Hg can beinduced with this method. The efficacy of the occlusion is evidenced byan immediate and sustained drop in the DBP measured in the tail artery.After approximately 10 minutes of induced spinal cord ischemia, theballoon is deflated, and the blood withdrawn from the carotid artery isreinfused. When the arterial blood pressure is stabilized (within about20-30 minutes after re-flow), the arterial lines are removed, and woundsare closed.

After the induction of spinal cord ischemia, the recovery of motorfunction is assessed in approximately 2-day intervals using a modified21-point open field locomotor scale (BBB scale)). Only animals with aBBB score of 0-4 are selected for the transplantation study forexperimental groups).

About 7-21 days following the ischemic lesion, spastic rats with BBBscore of 0-4 are anesthetized with 1.5-2% halothane in room air andplaced into a spinal unit apparatus. A partial laminectomy of Th11-L2vertebra is then performed. A glass capillary having a tip diameter of80-100 μm is connected to a pressure-controlled microinjector. Rats areinjected with 0.5 μl of cell suspension containing 5,000, 10,000, 15,000or 20,000 human neural stem/progenitor cells per injection. Each ratreceives a total of 6-8 injections on each side of the spinal cord (leftand right), evenly distributed between exposed L2-L6 segments. Thecenter of the injection is targeted into central gray matter (laminaeV-VII) (distance from the dorsal surface of the spinal cord at L3 level:1 mm). After implantation, the incision is cleaned with 3% H₂O₂ and apenicillin/streptomycin mixture and closed in 2 layers. Rats are thenallowed to recover.

Immunosuppressive treatment with FK-506 (Prograf; Fujisawa; 1 mg/kg;i.p.) is initiated in all animals about 3 days prior to spinaltransplantation. Following transplantation, the animals receiveimmunosuppressive treatment daily during the entire survival period.Immune rejection of these grafts can be effectively prevented withFK-506. The rats survive for approximately 2 or 7 weeks (n=5 for eachtime point).

At the end of the survival periods, rats are anesthetized withpentobarbital (40 mg/kg; i.p.) and transcardially perfused withheparinized saline for 1-2 min followed by 4% paraformaldehyde in 0.1Mphosphate buffer (PB). The spinal cords are dissected and postfixed inthe same fixative overnight at 4° C. After post-fixation, spinal cordtissue is cryoprotected in a graded sucrose solution (10, 20 and 30%)for a total of three days. Frozen coronal, parasagittal or horizontalspinal cord sections (10-30 μm) are then cut. For immunohistochemistry,free floating sections (30 μm) are placed in PBS, 0.1M (pH=7.4)containing 5% normal goat serum (NGS), 0.2% Triton X100 (TX), for twohours at room temperature to block the non-specific protein activity.This is followed by overnight incubation at 4° C. with various primaryhuman specific antibodies

After incubation with primary antibodies, sections are washed 3× in PBSand incubated with secondary goat anti rabbit or mouse antibodiesconjugated to fluorescent marker (Alexa 488 or 594; 4 μl/ml; MolecularProbes). All blocking and antibody preparations are made in 0.1MPBS/0.2% TX/5% NGS. For double labeling experiments, primary antibodiesfrom different species are applied simultaneously, followed byapplication of secondary antibodies conjugated to different fluorescentmarkers. In control experiments primary antibodies are omitted. Forgeneral nuclear staining, DAPI (3 μl/ml) is added to the final secondaryantibody solutions. After staining, sections are dried at roomtemperature and covered with Prolong anti-fade kit (Molecular Probes).

Slides are analyzed using a Leica Fluorescence microscope. Images(512×512 pixels) are captured with Olympus digital camera and processedby Adobe Photoshop 5.5 (Adobe Systems, Mountain View, Calif.). Toconfirm co-localization of different antibodies in double stainedsections images are captured with a DeltaVision deconvolution microscopesystem including a Photometrics CCD mounted on a Nikon microscope(Applied Precision, Inc.). In general, sixty optical sections spaced by0.1 or 0.2 μm are taken. Lenses used are 20×, 40× and 60× (NA 1.3). Thedata sets are deconvoluted and analyzed using SoftWorx software (AppliedPrecision, Inc) on a Silicon Graphics Octane workstation.

The total numbers of grafted neurons immunoreactive for the humannuclear NUMA antibody are estimated using stereological, unbiased andsystematic sampling. Each tenth, previously-stained section taken fromL2-L6 spinal segments is used for stereological quantification afterapplying fractionator sampling scheme. The optical images (1 μm thick)are obtained by Leica DMLB microscope using a 100× oil immersionobjective with numerical aperture 1.3. The optical images are capturedusing a digital camera (Olympus) and ImagePro software (MediaCybernetics) supplied with a StagePro-controlled motorized Z stage(Media Cybernetics). The total number of grafted cells are thencalculated by applying the fractionator formula N=Q×1/hsf×1/asf×1/ssf,where N is a total number of positive nuclei, Q is sum of cells counted,hsf is the height sampling fraction, asf is area sampling fraction andssf is slice sampling fraction.

To provide a three-dimensional reconstructed view of the grafted humanNSCs in the ischemic spinal cord, previously stored images taken fromserially cut spinal cords are used. On average, about 60-100 serialsections are used for three-dimensional reconstruction. In the firststep, a stack of serial images is opened using Ellipse software andaligned using the custom developed Allign module (VidiTo, SK). Thealignment process consists of defining two morphological referencepoints in all serial spinal cord images (the first point: center of thecentral canal; the second point: medial border of the dorsal horn), anda subsequent computer-processed alignment of all images. To identify theborders of the dorsal and ventral horns, lines are then draw on thestack of previously aligned images using Laminar Maps module (Ellipse).Finally, the stack of previously aligned and laminar maps-labeled imagesare used for three-dimensional reconstruction using 3-D constructor(Media Cybernetics).

Culture of human spinal NSCs on rat astrocytes for two to three weeksshowed a time dependent maturation and development of neuronal phenotypein the majority of the cultures. This is confirmed by staining withhuman-specific antibodies against NSE or MOC. Numerous neurons with awell developed axodendritic tree are also identified. The majority(85-90%) of NSE positive neurons are GABA positive. Expression ofsynaptophysin in axons and dendrites are observed in some MOC positiveneurons.

A robust survival of cells when grafted at twenty-one days afterischemic injury is seen. This is expressed as clearly-identifiedbilateral grafts immunoreactive for NUMA, MOC, or NSE, allhuman-specific antibodies. Analysis of horizontal sections taken fromgrafted spinal cord segments show a clear migration of NUMA positivecells between individual injection sites. The majority of NUMA-positivecells show co-localization with MOC immunoreactivity.

Double labeling of spinal cord sections with NUMA and GABA antibodiesanalyzed with confocal microscopy reveal an average of 25-35% GABApositive cells. A consistent expression of GABA-ergic phenotype is seenin all grafted animals at seven weeks of survival.

At the same time point (i.e. seven weeks after grafting),double-staining with synaptophysin and NUMA antibody show a densesynaptophysin-positive network within the grafts.

Only occasionally will NUMA-positive cells show co-localization withGFAP antibody. These cells are typically localized at the periphery ofthe grafts.

The stereological estimation of NUMA-positive cells shows an average of75,460±5,697 persisting grafted cells within individual grafts. Thisrepresents an average of 3-3.6 times more cells than originallyinjected. The cell-cycle antigen, Ki67, is a marker of actively mitoticcells. Staining of spinal cord sections at two or seven weeks aftergrafting show hKi67 immunoreactivity only at two weeks after grafting.Only occasionally (1-2 cells/10 sections) are cells Ki67-positive atseven weeks of survival. These results indicate that the grafted humanspinal NSCs and their progenies expand at a number equivalent to anaverage of about three cell-doublings for the initial two-week period,which then become post-mitotic and stably integrated.

Volume reconstruction of transplanted L3-L5 segments is performed byusing images taken from 40 μm-thick serial spinal cord sections stainedwith MOC antibody and DAPI (total number of 150-200 sections).Three-dimensional graft reconstruction shows a well recognizedrostrocaudally orientated MOC-positive implant distributed within therange of gray matter. As illustrated in FIG. 7 and FIG. 8, thefunctional effect of the human spinal NSCs are assessed by engraftingthe cells and measuring recovery motor benefit by BBB scoring.

With respect to the degree of behavioral recovery observed in thepresent study we have seen three principal groups after grafting. First,animals which displayed the most robust recovery and the ability to walk(BBB<16), second, animals which showed improvement in the activemobility of all 3 joints in the lower extremities but were not able tostand (BBB around 8), and, the third group in which animals which didn'tshow any recovery (i.e. non-responders). While the reason for thedifferences in responsiveness to the grafting is not clear we speculatethat a subtle differences in the graft positions with respect to thedysinhibited primary afferents and/or α-motoneurons can play a role.Furthermore it should be noted that animals survived only for 3 monthsin the present study. We speculate that a long term post-graftingsurvival and continuing physical rehabilitation will likely beassociated with a higher degree of functional recovery. Nonetheless, incontrast to treatment group no significant recovery was seen in anyanimal injected with medium only.

EXAMPLE 6 Treatment of Motor Neuron Diseases by Transplantation of HumanSpinal NSCs

The NSCs of the disclosed methods afford both clinical and biologicalbenefits that are powerful and significant. To this end, the disclosedmethods allow for the treatment of conditions both disseminatedthroughout the central nervous system, such as ALS, as well as localizedin a particular area as in spinal cord ischemia described above. In ALS,although grafting in the lumbar cord may omit other vital portions ofthe segmental motor apparatus, i.e. the cervical motor neuron columnresponsible for respiratory movements, the disclosed methods ofimplanting NSCs in the spinal cord facilitate the release of BDNF andGDNF and other factors from the transplanted cells into the CSF where abroader effect on host motor neurons throughout the cord can occur.

It has been surprisingly found that partial grafts of human NSCs intothe lumbar segments of the neurodegenerative spinal cord environmentsurvive, undergo extensive neuronal differentiation and promote motorneuron survival and function both at the site of implantation and atother locations. The NSCs significantly delay the onset of symptoms andextend the life of SOD1 G93A rats, a model of human ALS (amyotropiclateral sclerosis).

The SOD1 G93A rat represents a comprehensive model for theneuropathology and clinical symptoms of an especially aggressive form ofALS. [(Nagai et al., 2001; Howland et al., 2002)] NSCs from human fetalspinal cord can be grafted into the lumbar cord of SOD1 G93A rats andmice where extensive differentiation of the neurons occurs and where thedifferentiated neurons subsequently form synaptic contacts with hostneurons and express and release GDNF and BDNF. The rat SOD1 G93A model,for example, characterized by fulminant motor neuron disease can be usedto study and demonstrate the beneficial effects of NSCs in the disease.To this end, NSC grafts employed in the disclosed methods survive wellin a neurodegenerative environment and exert powerful clinical effects.At least a portion of these effects is related to the ability of thesegrafts to express and release motor neuron growth factors. Accordingly,the grafted NSCs of the disclosed methods delay the onset andprogression of the fulminant motor neuron disease and extend the lifespan of these animals by more than ten days, despite the restrictedgrafting schedule that was limited to the lumbar protuberance.

Human NSCs (NSI-566RSC) from spinal cord tissue of a post-mortem humanfetus of eight weeks gestational age are expanded in serum-free mediumcontaining fibroblast growth factor (FGF-2) for about 10-12 passagesprior to grafting (Johe et al., 1996). The fate of these cells arereliably traced with antibodies against human nuclear antigens (HNu)(Yan et al., 2003). All surgical procedures using these cells arecarried out according to protocols incorporated herein by referenceapproved by the Animal Care and Use Committee of the Johns HopkinsMedical Institutions using gas anesthesia (enflurane:oxygen:nitrousoxide=1:33:66) and aseptic methods.

Live or dead NSCs are grafted into the lumbar protuberance (L4 & L5) ofnine-week old (220-300 g) SOD1 G93A rats of mixed gender mounted on aKopf spinal stereotaxic unit under microscopic guidance. Dead cells areprepared by repetitive freezing and thawing before grafting. Cellsuspensions are delivered under aseptic conditions via approximatelyeight injections aimed at ventral horn on both sides of the ventral horn(5×10⁴ NSC per injection site, four injection sites per side) withpulled-beveled glass micro-pipettes connected, via silastic tubing, to10 μl Hamilton microsyringes. All rats receive FK-506 (1 mg/kg i.p.) toprevent immune rejection based on pilot data indicating that, inuntreated animals or animals receiving cyclosporin, graft survival doesnot exceed one month.

Rats are tested for motor strength and weight twice weekly. Motorstrength tests included the Basso, Beattie and Bresnahan (BBB) locomotorrating scale (Basso et al., 1995), and the inclined plane scale (Rivlinand Tator, 1977). For BBB score testing, animals are tested for about 4or 5 minutes in the open field. All locomotor performance is recordedand rated according to scale. For inclined plane testing, rats areplaced on the incline plane mat, and angle is adjusted to the maximalpoint at which their position can be stabilized for about 5 seconds.This angle is then recorded as the animal's inclined plane score. BBBand incline plane scores are analyzed by MANOVA followed by Fisher LSDpost hoc test. Disease onset is defined as the point at which bodyweight begins to decrease abruptly. Course of illness as an effect ofgraft type (live- or dead-cell graft) is analyzed by comparing age atdisease onset and age of death between the two groups (with students' ttest) as well as with Kaplan-Meier survival analysis followed bylong-rank test.

Rats are euthanized with perfusion-fixation when their BBB score (seebelow) is less than 3, a stage at which only one joint has movement orthere is no movement at all and the animal is considered moribund.

Tissues are prepared from animals perfused with 4% neutral-bufferedparaformaldehyde. The thoraco-lumbar spinal cord segments with attachedroots and lumbar nerves are further fixed by immersion in the samefixative for an additional four hours. Blocks containing the entiregrafted area plus 1 mm border above and below the block arecryoprotected and frozen for further processing. L3-S1 roots areprocessed separately as whole-mount preparations or after separatingrootlets with heat-coagulated tips of glass pipettes. Blocks aresectioned at the transverse or sagittal plane (35 μm). NSC survival anddifferentiation is studied with dual-label immunofluorescence thatcombines, in most cases, HNu, a human-specific marker, with anothercellular marker. and is performed as described in Yan et al., 2004.

A non-stereological method of counting total number of HNu(+) cells aswell as cells dually labeled with HNu and a phenotypic marker onrandomly selected high-power (100×) fields from our immunofluorescentpreparations is used to study NSC differentiation. One field in each ofsix sections spaced ˜1 mm apart through the grafting area is used fromeach animal. Numbers of HNu(+) and double-labeled profiles are pooledfrom all six fields counted from each case and grouped per experimentalprotocol. Average numbers of single and double-labeled cells aregenerated for each treatment group (n=6 per group).

To assess motor neuron survival in rats grafted with live or dead cells(n=4 of each), tissues from animals sacrificed at 128 days of age areevaluated. Every sixth section in the L3-S1 region from each animal issampled as per stereological requirements (Yan et al., 2004), andα-motor neurons, identified as multi-polar cells with a distinct nucleusand a soma diameter of >35 μm, are counted with the optical fractionatoras described in Yan et al., 2004. Differences between animals graftedwith live versus dead cells are analyzed with students' t test.

For ELISA determination of motor neurotrophic factors, CSF is sampledwith a 25 G syringe from the 4th ventricle of animals under gasanesthesia. Tissue samples containing the grafting sites and areasadjacent to those are dissected transversely from 1-mm-thick freshspinal cord slices. CSF or tissue samples are processed, and totalprotein is first measured as described in Sheng et al., 2003. Levels ofGDNF and BDNF are measured in CSF and spinal cord samples using theE-Max ImmunoAssay system (Promega, Madison, Wis.). TMB-chromogenabsorbance is read at 450 nm. Variance in concentrations among samplesfrom live grafts, areas adjacent to grafts and dead-cell grafts isanalyzed with one-way ANOVA followed by Tukey's Multiple Comparison posthoc test. Difference in CFS concentration between animals grafted withlive versus dead cells is analyzed with students' t test.

For western blotting of motor neurotrophic factors, Protein samples fromCSF or spinal cord prepared as for ELISA are electrophoresed withmolecular weight markers and transferred to nitrocellulose membranes.Blots are blocked in TBS, pH 7.4, containing 5% donkey serum and thenincubated in GDNF and BDNF antibodies (1:500; overnight, 4° C.) first,and then in HRP-linked donkey anti-goat IgG (for GDNF) and anti-rabbitIgG (for BDNF) (1:2000; Jackson ImmunoResearch) (1 hr, RT). Allantibodies are diluted in TBS containing 5% donkey serum. Blots aredeveloped with the SuperSignal Chemiluminescent Substrate (Pierce) andexposed to Kodak-XAR film (Eastman Kodak, Rochester, N.Y.). Blots arethen striped and re-blotted with β-actin antibody (1:500, Sigma) andHRP-linked donkey anti-mouse IgG (1:10000, Jackson ImmunoResearch).Immunoreactive bands are analyzed with Bio-Rad Quantity One software(Bio-Rad Laboratories, Hercules, Calif.). Band density ratios (GDNF orBDNF: β actin) are calculated per animal and group means are entered forstatistical analysis as in the case of ELISA experiments.

Human NSCs in the spinal cord of SOD1 G93A rats at 22 weekspost-graftingare identified by immunostaining with human-specific HNuantibody. HNu(+) cells are shown to survive in the ventral horn (A) andto stain, in their vast majority, with neuronal lineage markers such asthe microtubule-associated epitope TUJ-1. Human NSCs are identified bytheir human nuclear protein (HNu) signature and their phenotypic fatesare tracked with dual immunocytochemistry for HNu and epitopes specificfor neural precursor, neuronal and glial cells. At the end ofexperiments in SOD1 G93A rats, human NSCs show robust engraftment andexcellent long-term survival. A majority of HNu(+) cells (70.4±6.4%)differentiated into the neuronal lineage based on their co-localizationof TUJ-1. Approximately one-fifth (19.2±5.6%) of HNu(+) cellscolocalized with nestin and very few (1.3±0.9%) HNu(+) cells werepositive for GFAP.

The capacity of human NSCs to integrate within the host circuitry istested with perikaryal markers for graft/host cells and markersselective for either host or graft terminals. Sections are stained forHNu to establish graft origin, TUJ-1 to establish neuronaldifferentiation, and a monoclocal antibody for the pre-synaptic proteinBassoon (BSN) that recognizes rat and mouse epitopes, but not humanepitopes. A large number of HNu(+), TUJ-1(+) cells in parenchymallocations are found to be contacted by synaptic boutons of rat origin.

In confocal microscopy, NSC-derived neuronal cells with HNu (+) nucleusand TUJ-1(+) cytoplasm are contacted by rat terminals Conversely,preparations stained with TUJ-1 and human-specific synaptophysin revealdense terminal fields of small boutons apposed to host neurons,especially large and small motor neurons. A host motor neuron iscontacted by a large number of graft-derived boutons. Horizontalsections stained for HNu and human NF-70 show a large number ofgraft-derived axons leaving the graft on the left and coursingpreferentially along the white matter of the ventral funiculus.Cells/processes with ChAT immunoreactivity, are used to delineate grayfrom white matter in the ventral horn. A large number of axons labeledwith human-specific antibodies against the neurofilament epitope NF70are found in association with the grafting sites, evidence that manyhuman NSCs differentiate into projection neurons; these axons show apreference for the white matter of the ventral horn.

NSC grafts into the lumbar cord of SOD1 G93A rats prolong life span anddelay motor neuron death and disease onset and progression. Aprogression analysis of clinical and pathological measures in cases withlive-cell (L) and dead-cell (control, C) grafts is illustrated in FIG.9. Animals grafted with live NSCs showed a significantly increasedsurvival by both Kaplan-Meier and end-point analysis. A Kaplan-Meierplot (FIG. 9A) shows a significant separation between experimental andcontrol animals throughout the course of observation (P=0.0003). Timeplots of BBB open field and inclined plane test scores (FIG. 9B) show asignificantly slower progression in muscle weakness in animals graftedwith live NSCs compared to animals that had received dead NSCs.

The effect of NSCs on motor neuron survival in the lumbar protuberance(L3-S1) of Tg rats is examined in a small group of animals that receivelive or dead NSCs and euthanized at 128 days of age. The average lifespan for animals grafted with dead NSCs is 138 days, whereas ratsgrafted with live NSCs live for 149 days. Therefore, a significant11-day difference in life span occurs between experimental and controlrats (P=0.0005). An average time-to-disease-onset is 115 days foranimals that receive dead cells and 122 days for animals that aregrafted with live NSCs. A significant 7-day difference is observed intime-to-disease-onset between the two groups (P=0.0001).

Stereologically estimated numbers of α-motor neurons are 6,418 foranimals that receive live NSCs and 3,206 for rats that are grafted withdead NSCs, i.e. there are twice as many neurons in the lumbarprotuberance of experimental compared to control animals of the sameage. A difference of 3,212 cells in the lumbar protuberance between liveand dead NSC groups is observed (P=0.01) in a representativeexperimental and control rat at 128 days of age.

Potential mechanisms of neuroprotection afforded by human NSCs ondegenerating motor neurons include the expression and release of, twopeptides with classical trophic effects on mammalian motor neurons,[BDNF and GDNF] (Henderson et al., 1994; Koliatsos et al., 1993).Expression and release of GDNF and BDNF in the spinal cord of graftedSOD1 G93A rats is determined. Cord preparations and CSF samples areevaluated for BDNF and GDNF by Western blotting and ELISA. GDNFconcentrations in the parenchyma and CSF of rats grafted with live cells(L1 and L2) and animals grafted with dead cells (C) are determined byELISA. L1 represents concentrations through the grafting site, whereasL2 reflects concentrations in tissues one segment above or below.Variance among groups is significant and caused by a large differencebetween L1 or L2 and C groups.

Difference in CSF concentrations between experimental (live-cell, L) andcontrol (dead-cell, C) groups is also significant by t test. ELISAshowed a concentration of 0.912±0.050 pg/μg at the graft site and0.819±0.115 pg/μg one segment away in the spinal cord of liveNSC-grafted animals. In animals grafted with dead NSCs, theseconcentrations were 0.368±0.026 pg/μg in spinal cord segments containingthe graft. In the CSF, GDNF concentration was 0.027±0.012 pg/μl inexperimental and 0.006±0.002 pg/μl. These data demonstrate a three-foldincrease in the expression and release of GDNF in the cord and afive-fold increase in GDNF secretion in the CSF of animals with liveNSCs.

Western blotting also shows a higher normalized GDNF concentration inanimals grafted with live NSCs. GDNF Western blotting confirms the ELISApattern of increase by detecting a 16 kDa protein. Western blotting alsoshows a normalized GDNF density of 0.860±0.007 in live-cell grafts and0.708±0.052 in dead-cell grafts.

ELISA staining of BDNF in the parenchyma and CSF of experimental ratsand controls are determined. ELISA analysis shows a concentration of0.086±0.014 pg/μg at the graft site (L1) and 0.054±0.009 pg/μg onesegment away from graft in experimental animals (L2). In control rats,the BDNF concentration was 0.010±0.003 pg/μg in graft-containingsegments. The differences between experimental and control CSFconcentrations are significant. In the CSF, BDNF concentration is0.041±0.013 pg/μl in experimental animals and 0.010±0.008 pg/μl incontrols. These findings indicate an eight-fold increase in BDNFconcentration in the spinal cord and four-fold increase in the CSF ofexperimental animals. Together, the ELISA data suggest a more widespreadsecretion of GDNF compared to BDNF in animals grafted with live NSCs,especially in the CSF.

Immunocytochemistry also reveals that the vast majority of graftedHNu(+) cells are expressing GDNF. The source of the GDNF in animals withlive grafts is the grafted cells themselves. In the dually stainedpreparations for HNu (red) and GDNF (green) illustrate the abundance ofGDNF immunoreactivity within the cytoplasm of grafted NSCs. In animalsthat receive live grafts, there is intense GDNF immunoreactivity inround cytoplasmic structures resembling secretory vesicles within hostmotor neurons.

Confocal microscopy through a host motor neuron stained with GDNF andhuman synaptophysin (the latter to label graft-derived terminals)indicates the localization of GDNF in vesicular structures but not ingraft-derived terminals located on the surface of the illustrated hostmotor neuron. Sections stained with human synaptophysin antibodies (tolabel all graft terminals innervating host motor neurons) and GDNF showlack of any co-localization of the two proteins in boutons contactinghost motor neurons.

Rats with live grafts show an elaboration of pathways originating in thegraft and innervating structures in and around the central canal. Incontrast to the absence of GDNF protein in terminals contacting hostmotor neurons, the vast majority of NSC-derived axon terminalsinnervating the central canal co-localizes with GDNF immunoreactivity.Widespread co-localization of human synaptophysin and GDNF is observedwithin graft-derived terminals innervating ependymal cells in thecentral canal. These anatomical patterns indicate that GDNF is probablytaken up by host motor neuron terminals that innervate the graft viaretrograde transport and not delivered to these neurons viatrans-synaptic transfer (Rind et al., 2005)

The apparent resistance of grafted NCSs to the ongoing degenerativeprocess in the ventral horn of SOD1 G93A rats is especially promising.The survival and extensive differentiation of NSCs reported here is astrong indication that the inflammatory/excitotoxic signaling involvingmotor neurons harboring SOD1 G93A (Rothstein et al., 1992; Howland etal., 2002; Turner et al., 2005) has no evident toxicity on cells. Thisfactor alone raises optimism for future cellular strategies using graftsto restore motor function in degenerative motor neuron disease.

EXAMPLE 7 Treatment of Traumatic Spinal Cord Injury by Transplantationof Human Spinal Cord Neural Stem/Progenitor Cells. Treatment ofSyringomyelia

Expanded human spinal stem cells were injected into eitherimmunosuppressed, adult female Sprague-Dawley or immunodeficient,athymic nude rats. C₄₋₅ contusion lesions were produced in both groupsone month prior to transplantation. The graft recipients (n=24) survivedfrom 60-150 d post-transplantation. The human-derived NSCs formed largecellular aggregates consisting of neurons, astrocytes andoligodendrocytes. These grafts invariably and completely filled eachlesion. Immature-appearing, NeuN+/human nuclei+ neurons often comprised50% of the donor cell population. These neurons sent humanneurofilament+ processes through both gray and white matter fordistances of at least 2 cm from the graft site. Intense human-specificsynaptophysin immunoreactivity was noted in proximity of both host andgraft neurons, and seemingly unreactive, GFAP+ cells were juxtaposedwith the donor neurons. Further, these transplants supported the growthof TH+ and 5HT+ fibers that appeared to arise from host sources. Thisline of NSC's thus appears to possess primary fetal CNS-like qualitiesfavorable to intraspinal gray matter repair.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the disclosed methods andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

1. A method of culturing human neural stem cells, said methodcomprising: providing human neural stem cells; incubating a culturevessel with an about 0.1 μg/mL to about 1 mg/mL solution ofpoly-D-lysine for about 5 minutes to about 3 hours; washing and dryingthe culture vessel to form a pre-coated culture vessel; seeding theneural stem cells in said pre-coated culture vessel in absence of serum;adding a fibronectin solution and at least one mitogen to the pre-coatedculture vessel and culturing the neural stem cells in absence of serum;further culturing the neural stem cells with at least one mitogen; andpassaging the cultured neural stem cells prior to confluence.
 2. Themethod of claim 1, wherein the mitogen includes bFGF.
 3. The method ofclaim 1, wherein the mitogen includes EGF.
 4. The method of claim 1,wherein the mitogen includes TGF-alpha.
 5. The method of claim 1,wherein the mitogen includes aFGF.
 6. The method of claim 1 wherein thesolution of poly-D-lysine has a poly-D-lysine concentration of about 100μg/mL.
 7. The method of claim 1 wherein the step of incubating a culturevessel with a solution of poly-D-lysine is performed for about 1 hour.8. The method of claim 1 wherein the fibronectin solution has afibronectin concentration of about 1 μg/mL.
 9. The method of claim 1wherein the fibronectin solution and the at least one mitogen are addedto the pre-coated culture vessel substantially at the time of seeding.10. The method of claim 1 wherein the provided neural stem cells arederived from human embryonic stem cells.
 11. The method of claim 1wherein the provided neural stem cells are from a central nervous systemtissue.
 12. The method of claim 11 wherein the central nervous systemtissue comprises spinal cord tissue.
 13. The method of claim 12 whereinthe spinal cord tissue is obtained from a post-mortem fetus having agestational age of about 6.5 to about 20 weeks.